Heme oxygenase, which exists in inducible (HO-1) and constitutive (HO-2) forms, oxidizes heme to CO, biliverdin, and ferrous iron (Figure 1). The electrons required for catalytic turnover of the enzyme are provided, in mammalian systems, by NADPH-cytochrome P450 reductase. The enzyme is of clinical interest for several reasons. First, biliverdin is the sole precursor of bilirubin and heme oxygenase is a potential target for the control of hyperbilirubinemia in neonatal children and in a number of hereditary disorders. Second, the biliverdin and bilirubin produced by the enzyme are potent antioxidants and appear to be involved in a manifold of protective physiological situations, including the prevention of atherosclerosis. Third, the iron released by the enzyme is required for maintenance of iron homeostasis. In the absence of heme oxygenase, the iron distribution in the body is flawed. Finally, but most intriguingly, the carbon monoxide produced by the constitutive form of heme oxygenase appears to act, in at least some situations, as a messenger akin to nitric oxide.
Figure 1. Reaction intermediates in the conversion of heme to biliverdin and CO by heme oxygenase.
Heme oxygenase is a membrane-bound, intractable enzyme but we circumvented the associated problems by expressing a truncated, soluble form of the protein in E. coli. The expressed protein retains full catalytic activity. In collaboration with the group of Thomas Poulos at the University of California at Irvine, we have determined the crystal structure of the truncated human protein
We demonstrated earlier that catalytic turnover of the enzyme can be supported by H2O2 rather than cytochrome P450 reductase/NADPH/O2. The reaction supported by H2O2 gives verdoheme, an intermediate in the normal reaction sequence that is converted to biliverdin by a reaction that requires NADPH and oxygen. Thus, H2O2 can replace oxygen and reducing equivalents in a-meso-hydroxylation of the heme. In contrast, the reaction with alkylhydroperoxides other than ethylhydroperoxide produces a ferryl species analogous to that obtained with the peroxidases. Ethylhydroperoxide reacts with heme oxygenase to give a-meso-ethoxyheme. The reaction with ethylhydroperoxide thus proceeds exactly as expected for the first step in the normal reaction. The results indicate that a-meso-hydroxylation involves electrophilic addition of the protonated intermediate Fe-O-OH to the porphyrin ring.
The four regioisomeric meso-methyl- and meso-formyl derivatives of mesoheme were synthesized and their oxidation by heme oxygenase examined. Surprisingly, despite the blocking methyl group, alpha-meso-methylmesoheme is oxidized to alpha-mesobiliverdin. No CO is formed in the reaction. Even more surprising is the fact that gamma-meso-methylmesoheme is oxidized at the blocked gamma- rather than unblocked alpha-meso position to give gamma-mesobiliverdin. These results argue for an electrophilic alpha-meso-hydroxylation mechanism and suggest that the normal alpha-meso regiospecificity is determined, in part, by electronic mechanisms. Additional results show that electron donating groups favor, and electron withdrawing groups disfavor, oxidation of a given meso-carbon.
Mutations of glycine residues (Gly38, Gly42) at the ends of a helix that regulates binding of the heme and release of biliverdin impair or suppresse heme oxygenase activity and convert the enzyme into a peroxidase. The same is observed if an aspartic residue (Asp140) that is probably involved in hydrogen bonding to the catalytic species is mutated. The mechanism of the enzyme, and the activities and properties of other mutants, are under continuing investigation.
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